Figure 3

Interferon responsiveness of mouse and human p47 (IRG) GTPase. (a) Interferon (IFN)-γ responsiveness of eight new mouse Irg genes. Inducibility of eight further Irg genes (also see Boehm and coworkers [13]) in L929 fibroblasts induced for 24 hours with IFN-γ, demonstrated by RT-PCR. D refers to a positive control genomic DNA template; O refers to a negative control of the same genomic template after DNAse1 treatment; and + and - refer to RT-PCR on DNAse1-treated RNA templates from IFN-γ-induced and IFN-γ-noninduced cells, respectively. The sibling genes of the Irgb series could not be individually amplified because of their close sequence similarity. The identities of the amplified genes responding to interferon induction, indicated by vertical arrows, were subsequently established by sequencing of multiple clones from the PCR product. (b) Irgc is not induced by interferon or infection but is constitutively expressed in testis. (i, left) Mouse L929 fibroblasts were induced for 24 hours with IFN-β or IFN-γ or left uninduced (-). Irgc could not be detected by RT-PCR even after 50 amplification cycles in L929 cells. Irga2 after 50 cycles was used as a positive control for the interferon-induced L929 RNA. RNA from mouse testis provided a positive control for Irgc. (i, right) RT-PCR for Irgc and Irga2 (50 and 30 amplification cycles respectively) on RNA from tissues of uninfected mice (-) or mice infected 24 hours previously with Listeria monocytogenes (+). Irga2 was induced in all tissues and Irgc in none. RNA from mouse testis provided a positive control for Irgc, which is detected after 50 cycles. Testis expression of Irga2 was barely detected after 30 cycles (compare with i, left, showing Irga2 in testis after 50 cycles). (Panel ii, left) Human IRGC is not induced by 24 hours of stimulation with IFN-β or IFN-γ in human cell lines (induction of GBP-1 [accession number P32455] was assayed as a positive control) and (Panel ii, right) is constitutively expressed only in human testis. GAPDH was used as control.